The main objective of this research proposal is to investigate neuropharmacologically the cellular mechanisms of arousal and sleep with a special emphasis on the pathogenesis of narcolepsy. Hypocretins/orexins, newly discovered peptide neurotransmitters, and their receptors will be studied. Hypocretins/orexins and their receptors play a role in narcolepsy and sleep disorders and in the regulations of food intake. Currently little is known about their physiological effects on brain neurons. Previous studies have shown that lack of excitation produced by hypocretins/orexins through hypocretin2/orexin2 receptors in the brain nuclei is the central component of narcolepsy. Proposed projects focus on further elucidating their actions at the cellular and molecular level by using dissociated primary cultures of rat and mouse brain nuclei that are involved in regulating arousal and sleep. The specific projects are: (1) to elucidate hypocretin/orexin effects on histaminergic neurons in the tuberomammillary nucleus which are rich in hypocretin2/orexin2 receptors, (2) to determine the transmitter effects on noradrenergic neurons in the locus coeruleus which are rich in hypocretin1/orexin 1 receptors, and (3) to investigate hypocretin/orexin effects on cholinergic neurons in the nucleus basalis of Meynert. In addition, a heterologous system (HEK293A) which is transfected with each type of hypocretin/orexin receptor will be used. All of these projects emphasize elucidating signal transduction mechanisms of hypocretins/orexins, and determining the identity and roles of G proteins. Electrophysiological techniques (patchclamp) and pharmacological techniques combined with molecular biological methods will be used. These projects are important for deepening the understanding of narcolepsy and other sleep disorders. The knowledge obtained is essential in developing more effective treatments for narcolepsy and other sleep disorders. This research on the sleep-arousal centers of the brain will contribute in helping those who suffer sleep disorders.